Genetic Code & Translation

Triplets Triplets of nucleotide bases are the smallest units of uniform length that can code for all the amino acid (20) Three consecutive bases specifiy a particular amino acid During transcription, the template strand of DNA provides the template for ordering the nucleotide sequence in the RNA transcript RNA transcript provides the template for the ordering of the amono acids

Complementarity RNA molecule is synthesized according to base pairing rules –Except Uracil is complementary to adenine RNA is synthesized in an antiparallel direction to the template strand of DNA mRNA base triplets are called codons The codons are written in the 5` to 3` direction

During translation –Sequence of codons along an mRNA is translated into a sequence of amino acids Codons are read 5` to 3` Each codon specifies which one of the 20 amino acids will be incorporated into the corresponding position along the polypeptide

Marshall Nirenberg Determined the first codon to amino acid match –UUU=phenylalanine He created an artificial mRNA entirely of uracils Added it to a test tube with amino acids, ribosomes and other components necessary for protein synthesis The “poly=U” mRNA translated into a polypeptide consisting only of phenylalanine By the mid 1960’s the entire code was deciphered

Codons There are 64 codons –3 do not code for any amino acids, these are STOP codons (UGA, UAG, UAA) Signaling the end of translation –1 codes for methinione, but is also the “start” codon for translation (AUG) –All other code for a single amino acid There is redundancy –Several codons may specify for the same amino acid –No codons specify more than one amino acid

Reading Frame Specification of the correct starting point Subsequent codons are read in groups of 3 nucleotides –Non-overlapping 3 letter words Each codon=specific amino acid

Universal The genetic code is shared by organisms from bacteria to the most complex plants and animals Genes can be transplanted into a different species and transcribed and translated –Permits bacteria to be programmed to synthesize certain human proteins Biotechnology at work

Exceptions There are a few exceptions to the universality of the gene code –Certain unicellular eukaryotes –Organelle genes of some species –Some prokaryotes can translate stop codons into one of two amino acids not found in most organisms

Evolutionary Significance A language shared by all living things must have arose very early in evolution

Concept 17.4 Translation is the RNA- directed synthesis of a polypeptide

tRNA In translation, a cell “interprets” a series of codons along an mRNA to build a polypeptide tRNA is the interpreter –Transfers amino acids from the cytoplasm to a ribosome –The 20 amino acids are either synthesized from scratch or taken up from surrounding solution The ribosome adds each amino acid carried by tRNA to the growing end of the polypeptide chain

Codons & Anticodons Each tRNA carries a specific amino acid at one end and has a specific nucleotide triplet sequence (anticodon) at the other end During translation, each type of tRNA links with an mRNA codon with the appropriate amino acid The anticodon form complementary base pairs with the codon on mRNA –Codon=UUU –Anticodon=AAA –Amino acid=phenylalanine

Translator tRNA acts as a translator –Reads the nucleic acid word (codon on mRNA) –Translate it into protein word (amino acid) Codon by codon, the tRNA deposits amino acids in the prescribed order The ribosome then joins them into a polypeptide chain tRNA is transcribed from DNA template tRNA can be used repeatedly

tRNA structure Strand of ~80 nucleotides that folds back on itself to form a three dimensional structure –loop for the anticodon –3` attachment site for an amino acid If there needs to be a perfect match between codon and anticodon, then how many anticodons are there? 45 The anticodons of some tRNA recognize more than one codon

Wobble 45 anticodons are possible because the rules for pairing the third base of the codon and anticodon are relaxed –At the wobble position U on anticodon can bind with A or G on the third position of the codon –Wobble explains why codons for a given amino acid can differ in their third base, but usually not in the others.

Synthetase Each amino acid is joined to the correct tRNA by aminoacyl-tRNA synthetase 20 different synthetases match the 20 different amino acids Each tRNA has an active site for a specific tRNA-amino acid combination Synthetase catalyzes a covalent bond between tRNA and amino acid –ATP hydrolysis drives the reaction –Results in aminoacyl-tRNA (activated amino acid)

Ribosomes Ribosomes facilitate the specific coupling of the tRNA anticodons and mRNA codons Large and small subunits Each subunit is composed of proteins and rRNA (ribosomal rRNA) –Most abundant RNA in cell

Ribosome formation Subunits are made in the nucleolus rRNA genes are transcribed to rRNA in nucleus Subunits exit nucleus via nuclear pores Large and small subunits join to form a functional ribosome ONLY when they attach to an mRNA

Prokaryotic ribosomes Very similar to eukaryotic ribosome Different enough to be paralyzed by certain antibiotics (tetracycline)

Binding sites Each ribosome has a binding site for mRNA and 3 binding sites for tRNA –P site=holds the tRNA carrying the growing polypeptide chain –A site=holds the tRNA with the next amino acid to be added to the chain –E site=discharged tRNA leave the ribosome

Ribosome Holds the tRNA and mRNA in close proximity Positions the new amino acid for addition to the carboxyl end of the peptide Catalyzes the formation of a peptide bond As the polypeptide elongates, it passes through an exit tunnel in the ribosomal large unit and is released to the cytosol rRNA, not proteins, carry out the ribosomal functions

3 stages Initiation Elongation Termination All three phases require protein factors that aid in the translation process Initiation and elongation require energy provided by the hydrolysis of GTP –Similar to ATP

Initiation Brings together mRNA and a tRNA (carrying the first amino acid) and the two ribosomal subunits –1 st : a small subunit binds with mRNA and a initiator tRNA (methionine) –2 nd : the small subunit moves downstream along the mRNA until it reaches the start codon (AUG) This established the reading frame for the mRNA –The initiator tRNA hydrogen bonds with the start codon –3 rd : initiation factors (proteins) bring in the large subunit so that the initiator tRNA occupies the P site

Elongation Involves several elongation factors (proteins) Three step cycle as each amino acid is added –Codon recognition An elongation factor assists hydrogen bonding between the mRNA codon under the A site with the corresponding anticodon of the tRNA carrying the appropriate amino acid

1 st stage of elongation Codon recognition An elongation factor assists hydrogen bonding between the mRNA codon under the A site with the corresponding anticodon of the tRNA carrying the appropriate amino acid

2 nd stage of elongation Peptide bond formation –An rRNA molecule catalyzes the formation of a peptide bond between the polypeptide in the P site with the new amino acid in the A site –Separates the tRNA at the P site from the growing polypeptide chain and transfers the chain, to the tRNA in the A site

3 rd stage of elongation Translocation –Ribosome moves the tRNA with the attached polypeptide from the A site to the P site –Because the mRNA is still bonded to the anticodon, the mRNA moves along with the tRNA –The next codon is now available at the A site –The tRNA that was in the P site is now in the E site and leaves the ribosome –Translocation is fueled by GTP –This continues codon by codon until the polypeptide chain is completed

Termination Occurs when one of the three stop codon reaches the A site –A release factor binds to the stop codon and hydrolyzes the bond between the polypeptide and its tRNA in the P site –The frees the polypeptide and the translocation complex disassembles

Polyribosomes Typically a single mRNA is used to make many copies of a polypeptide simultaneously

Lets go to the video A ribosome requires less than one minute to translate an average sized mRNA into a polypeptide During and after synthesis a polypeptide coils and folds to its three dimensional shape –Primary structure (order of amino acids) determines secondary and tertiary structure –Chaperone proteins may aid in correct proteins

Posttranslational modifications Proteins may require additional modifications after translation –Addition of sugars, lipids, or phosphate groups to amino acids –Removal of some amino acids –Cleavage of whole polypeptide chains –Joining of two or more polypeptide chains

Signal peptides Target some eukaryotic polypeptides to specific destinations in the cell –Two populations of ribosomes: Identical in structure, location depends on the type of protein they are synthesizing Free –Suspended in the cytosol –Synthesize proteins that reside in the cytosol Bound –Attached to the cytosolic side of the ER –Synthesize proteins of the endomembrane system and proteins that will be secreted from the cell

Signal peptide Translation in all ribosomes begins in the cytosol Polypeptides destined for the endomembrane system or export has a specific signal peptide region at or near the leading end (~20 amino acids) SRP (signal recognition particle) binds to the signal peptide and attaches it and its ribosome to a receptor protein in the ER membrane –Protein-RNA complex

After binding After the ribosome is bound to the ER the SRP leaves and protein synthesis resumes The polypeptide passes through a protein pore into the cisternal space The signal polypeptide is usually cleaved by an enzyme during final processing of the protein

Secretory proteins are completely released into the cisternal space Membrane proteins remain partially embedded in the ER membrane Other signal peptides are used to target polypeptides to mitochondria, chloroplasts, the nucleus and other non-endomembrane organelles –Translation is completed in the cytosol before importation to the organelle –They are coded with a “zip” code that directs its delivery to the correct organelle

Concept 17.5 RNA plays multiple roles in the cell

Types of RNA Involved directly in protein synthesis –mRNA –tRNA –rRNA –snRNA –SRP RNA

Types of RNA Small nucleolar RNA (snoRNA) –processing of pre-rRNA transcripts in the nuceolus A process necessary for ribosome formation Small interfering RNA (siRNA) & microRNA (miRNA) –Small, single stranded and double stranded RNA molecules play an important role regulating which genes get expressed

Why so many RNAs Its ability to form hydrogen bonds with other nucleic acids (DNA or RNA) Its ability to form a specific three dimensional shape by forming hydrogen bonds between bases in different parts of itself DNA may be the genetic material of all living cells, but RNA is much more versatile –Structural –Functional –catalytic

Concept 17.6 Comparing gene expression in prokaryotes and eukaryotes reveals key differences

Differences Different RNA polymerases Eukaryotic RNA polymerases require transcription factors Differences in termination Ribosomal structure differences Prokaryotes can transcribe and translate a gene at the same time In Eukaryotes, extensive RNA processing occurs Eukaryotes have complicated mechanisms for targeting proteins to the appropriate organelle

Concept 17.7 Point mutations can affect protein structure and function

Mutations Changes in the genetic material of a cell (or virus) Large scale mutations in which long segments of DNA are affected –Translocations –Duplications –Inversions Chemical change in just one base pair of genes –Point mutation

Point mutations Occurring in gametes or early in development may be transmitted to future generations –Sickle cell anemia Point mutation of one base (T to A in the DNA) in one of the polypeptides for hemoglobin

Base-Pair substitution Replacement of a pair of complementary nucleotides with another nucleotide pair –Silent mutation Altered mutation still code for the same amino acid because of the 3 rd base redundancy Other may switch one amino acid to another with similar properties May occur in a region where the exact amino acid sequence is not essential for function –Detectable change in a protein Usually detrimental May occasionally lead to an improved protein Changes in amino acids at crucial sites (active sites) are likely to impact function

Other point mutations Missense mutations –Still code for an amino acid, but a different one Nonsense mutation –Changes an amino acid into a stop codon, –Nearly always leads to a nonfunctional protein

Insertions & Deletions Additions or losses of nucleotide pairs in a gene Have a greater effect on the resulting protein than substitutions do Unless they occur in multiples of three they cause a frameshift mutation All nucleotides downstream of the deletion or insertion will be improperly grouped into codons Result is missense, ending sooner or later in nonsense-premature termination

Mutations Errors can occur during DNA replication, DNA repair, or DNA recombination Spontaneous mutations Rough estimates suggest that 1 nucleotide in every is altered and inherited by daughter cells

Mutagens Physical agents –High energy radiation –X-rays –Ultraviolet rays Chemical agents –Base analogues which may substitute into the DNA which pair incorrectly during DNA replication –Some interfere with DNA replication by inserting into the DNA and distorting the double helix –Others cause chemical changes in bases that change their pairing properties

Testing Mutagen effects Researchers have developed various methods to test mutagenic activity of different chemicals Preliminary screen of chemicals to identify carcinogens Most carcinogens are mutagenic Most mutagens are carcinogenic